Abstract Neutrophil extracellular traps (NETs) are extracellular fibrillary structures of chromatin filaments coated with histones, proteases and granular and cytosolic proteins released by neutrophils as an antimicrobial mechanism that `traps' and kills bacteria. Cumulative research reveals that NETs' antimicrobial killing properties can also induce tissue injury when dysregulated. Hence, NETs are increasingly recognized as a culprit-driver in the pathogenesis of multiple major diseases ? acute respiratory distress syndromes (ARDS), acute coronary syndromes (ACS), multi-organ failure (MOF) in ARDS, and sepsis ? where durable breakthrough therapies are lacking, despite significant research. Regardless of the disease, the fact that NETs are the common culprit in diverse and pathogenically disparate diseases argues the importance and high-value priority of targeting NETs. We hypothesize that successful neutralization and dismantlement of intravascular NETs will stop NET- driven endothelial injury at pulmonary vascular-alveolar barrier injury sites in acute respiratory distress syndrome (ARDS). To overcome the concomitant biological and biophysical barriers to dismantling NETs and neutralizing NET-driven tissue injury, a multi-pronged therapeutic is needed. We will therefore develop a novel therapeutic that comprises: 1) a highly specific, humanized hinge-stabilized S228P IgG4 antibody that targets the dual endothelin1/signal peptideVEGF receptor (DEspR) detected on NETosing neutrophils ? anti-DEspR- humab; and, 2) DNase1 conjugated to a tripeptide linker that is cleaved by cathepsin G (cg). Release of the DNase1 by cathepsin G cleavage at the NET site will facilitate NET dismantlement and serve as a substrate decoy for cathepsin G reactivity, thus minimizing its direct endothelial injury activity. This proposed targeted enzymatic bioconjugate is enabled by a novel method of stoichiometric, site-specific conjugation to antibodies ? i.e., the NanoZip which utilizes the supramolecular assembly of coiled coils (SMACC) to achieve selective, specific coupling of two DNase1 enzymes to the C-terminus of an antibody. The specific aims of this two-year R21 proposal are: Aim 1. Prepare the antiDEspR-humab-cg-peptide-DNase1 therapeutic prototype (DESPRnase1) and evaluate structural stability and dose-dependent release of DNase1 by cathepsin G in basal plasma conditions, and in the presence of low pH and high ROS milieus present in ARDS. Aim 2. Determine whether DESPRnase1 targets and binds to DEspR+ NETs, dismantles DEspR+ NETs without complement activation, and/or serves as substrate-decoys to attenuate NETs' cathepsin G-induced injury of human endothelial cells ex vivo. This R21 will develop a prototype bio[nano]conjugate as a breakthrough therapeutic to dismantle NETs and stop the vicious cycle of endothelial injury in ARDS. Notably, efficacy in ARDS will open the door to potential applications in ACS and other indications.